This disclosure relates generally to the field of systems and methods for determining whether an agent (e.g., bacterium) is present in a biological or clinical sample such as blood or urine.
Instruments currently exist on the market in the U.S. that detect the growth and therefore the presence of a microorganism in a blood sample. One such instrument is the BacT/ALERT 3D instrument of the present assignee bioMérieux, Inc. The instrument receives a blood culture bottle containing a blood sample, e.g., from a human patient. The instrument incubates the bottle. Periodically during incubation an optical detection unit in the incubator analyzes a colorimetric sensor incorporated into the bottle. The reflection measurements obtained by the detection unit are used to detect whether microbial growth has occurred within the bottle. The optical detection unit, specimen containers and sensors are described in the patent literature, see U.S. Pat. Nos. 4,945,060; 5,094,955; 5,162,229; 5,164,796; 5,217,876; 5,795,773; and 5,856,175, the entire content of each of which is incorporated by reference herein. U.S. Pat. Nos. 5,856,175 and 5,164,796 describe methods for determining whether microbial growth is occurring with a sample container.
The performance of the positive bottle detection algorithm of the BacT/ALERT instrument is considered commercially acceptable. However, it has several shortcomings. First, the time to detection (TTD) appears to be delayed in some cases when the TTD is compared to a visual inspection of the reflectance curve. In other words, the detection occurs later in the exponential growth phase (see FIG. 2 and the description that follows) than what would be expected. Second, false positive results are known to occur as a result of events such as temperature effects from loading relatively cold bottles, re-loading bottles in different cells in the incubator, and bottles being moved within the same cell. Third, false negative results are known to occur in the case of a delayed loading of bottles. A false negative result is observed when only the upper portion of the exponential phase is detected or the stationary phase is not at a reflectance level high enough to trigger the initial reflectance value positive threshold. Fourth, the algorithm logic is considered complex, difficult to understand, and difficult to maintain.
Other prior art of interest relating generally to the detection of microorganisms in a biological sample includes the following patents: U.S. Pat. Nos. 5,770,394, 5,518,923; 5,498,543, 5,432,061, 5,371,016, 5,397,709, 5,344,417, 5,374,264, 6,709,857; and 7,211,430. The following patent documents are also of potential interest: U.S. Pat. Nos. 7,991,558; 7,668,663; US 2009/0119020; US 2011/0029252; US 2011/0208432; US 2009/0287754 and US 2010/0070190.
In detection instruments such as the BacT/ALERT 3D and similar instruments, once the blood culture bottle has been tested positive for microorganism presence, it is difficult to obtain a high level of characterization of the microbial agent, or identification of the species of the microbial agent, due to the interference of blood components and artifacts of the disposable system (e.g., bottle) containing the sample. Therefore, current methods use a bottle or other suitable disposable container and a related instrument for natural growth and detection of a microorganism in the sample, as described above. Once the instrument indicates that the bottle is positive for presence of a microbial agent, according to current methods the “positive” bottle is manually retrieved from the instrument and a portion of the sample is manually removed from the bottle and cultured on an agar plate. The plate is manually placed in an incubator and periodically inspected for growth of a subculture of the microorganism. After the subculture has grown sufficiently, a sample of the culture is taken from the plate and placed in a test tube. The test tube is then introduced into yet another instrument for identification testing via a disposable test sample card having a multitude of individual wells. The disposable test cards are known in the patent literature, see e.g., U.S. Pat. Nos. 4,118,280, 3,963,355, 4,018,652; 4,116,775 and 4,038,151, 5,609,828, 5,746,980, 5,766,553, 5,843,380, 5,869,005, 5,916,812, 5,932,177, 5,951,952, and 6,045,758, the entire content of which is incorporated by reference herein.
The test sample card is then processed in an analytical instrument known in the art as the VITEK 2 instrument of the assignee. The VITEK 2 instrument incubates and periodically reads the wells of the test sample card with a reader unit. Growth of the sample in one or more of the wells of the cards results in identification of the microbial agent. The VITEK 2 instrument is described in the patent literature, see e.g., U.S. Pat. Nos. 5,762,873 and 6,086,824, the content of which is incorporated by reference herein.
This entire process from the time of introducing the sample into the blood collection bottle to culture, detection of microorganism presence, and then identification of the microorganism by the VITEK 2 instrument typically takes 2-5 days. The identification steps alone, occurring after positive bottle detection, typically occupy 1-3 of these days.
Substantial, and potentially life-saving, clinical benefits for a patient are possible if the time it takes for detection and identification of a microbial agent in a blood sample and reporting the results to a clinician could be reduced from the current 2-5 days to less than one day.
In a related application of the applicant's assignee, published as U.S. 2011/0281291, methods for identifying a microbial agent in a specimen container are disclosed. In the present disclosure, methods are disclosed for detecting whether microbial growth in a sample container is occurring, thereby indicating that an agent is present in the sample. The methods reduce the time required to make this initial determination. Because the initial determination is made earlier, the second step of identifying the agent (such as described in U.S. 2011/0281291) can be initiated earlier than otherwise possible. This invention thus contributes to an overall reduction of the amount of time needed for detection and identification of the microbial agent. Moreover, the methods of this disclosure overcome the deficiencies of current detection algorithms.